XBB.1.16‐RBD‐based trimeric protein vaccine can effectively inhibit XBB.1.16‐included XBB subvariant infection

Abstract The newly identified XBB.1.16‐containing sublineages, including XBB.1.5, have become the prevailing severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) variant in circulation. Unlike previous Omicron XBB variants (e.g., XBB.1.5 and XBB.1.9) harboring the F486P substitution, XBB.1.16 also carries a T478R substitution in the receptor‐binding domain (RBD). Numerous researchers have delved into the high transmissibility and immune evasion of XBB.1.16 subvariant. Therefore, developing a new vaccine targeting XBB.1.16, including variants of concern (VOCs), is paramount. In our study, we engineered a recombinant protein by directly linking the S‐RBD sequence of the XBB.1.16 strain of SARS‐CoV‐2 to the sequences of two heptad repeat sequences (HR1 and HR2) from the SARS‐CoV‐2 S2 subunit. Named the recombinant RBDXBB.1.16‐HR/trimeric protein, this fusion protein autonomously assembles into a trimer. Combined with an MF59‐like adjuvant, the RBDXBB.1.16‐HR vaccine induces a robust humoral immune response characterized by high titers of neutralizing antibodies against variant pseudovirus and authentic VOCs and cellular immune responses. Additionally, a fourth heterologous RBDXBB.1.16‐HR vaccine enhances both humoral and cellular immune response elicited by three‐dose mRNA vaccines. These findings demonstrate that the recombinant RBDXBB.1.16‐HR protein, featuring the new T478R mutation, effectively induces solid neutralizing antibodies to combat newly emerged XBB variants.


INTRODUCTION
Earlier in 2023, the XBB.1.16variant emerged in India before spreading to other Western and Asian countries, marking a significant development in the trajectory of COVID-19. 1 Within a mere 4 weeks, its prevalence surged, leading to its classification as a variant of interest (VOI) on April 17, 2023. 2 Stemming from the XBB.1.5subvariant and characterized by the F486P substitution in the receptorbinding domain (RBD), XBB.1.16also harbors a T478R substitution in the RBD.Extensive research has investigated its high transmissibility and immune evasiveness, with its adequate reproductive number being 1.22-and 1.13-fold higher than XBB.1 and XBB.1.5,respectively. 3,4espite a lower binding affinity of XBB.1.16RBD to the human angiotensin-converting enzyme 2 (ACE2) receptor compared to XBB.1.5, the profound immune evasion of XBB.1.16was similar to XBB.1.5. 4 These findings highlight the urgent need to develop a new vaccine targeting newly emerged variants like XBB. 1.16.Additionally, the World Health Organization (WHO) supports creating monovalent vaccines targeting only one strain, underscoring the urgency of developing a monovalent vaccine to combat newly emerged variants and prevent immune escape. 5eople in many countries have already been immunized with the mRNA COVID-19 vaccine (mRNA-1273 and mRNA BNT162b2). 6,7However, despite their efficacy at preventing infection by the ancestral strain, their antibody response to BA.4/5-included Omicron sublineages has been limited. 8To address the challenge, bivalent COVID-19 vaccines were designed, combining the spike of Wuhan-1 with either BA.1 (mRNA-1273.214) or BA.4/5 (mRNA-1273.222).These bivalent COVID-19 vaccines have demonstrated the ability to broadly neutralize antibodies and enhance protection against BA.4/5 viruses in mice and clinical trials. 9Moreover, booster vaccines of both mRNA-1273.214and mRNA-1273.222have shown promise in providing additional protection against BA.4/5-included Omicron sublineages, [10][11][12] with mRNA-1273.222being particularly effective in preventing COVID-19-related outpatient visits and hospitalizations in older adults (≥65). 13ubsequently, the food and drug administration (FDA) authorized using the mRNA-1273.222vaccine as a booster dose.Furthermore, research has investigated the efficacy of using the RBD XBB.1.16 -HR vaccine as a heterologous booster.
Furthermore, the potential of the RBD XBB.1.16-HR vaccine as a heterologous sequential booster following mRNA vaccine priming has been explored.After three doses of mRNA vaccine, a fourth dose of the heterologous RBD XBB.1.16-HR boosting vaccine has shown potential to enhance vaccine-induced humoral and cellular immunity.In conclusion, studies suggest that the RBD XBB.1.16-HR vaccine, incorporating the new T478R mutation, could induce neutralizing antibodies capable of combating newly emerged XBB subvariants.

Construction and characterization of the recombinant RBD XBB.1.16 -HR protein
The construct design is illustrated in Figure 1A.We essentially engineered an RBD XBB.1.16-HR protein compromising an RBD (320-545 aa) sequence derived from the XBB.1.16Omicron subvariant, along with HR1 and an HR2 sequences derived from the SARS-CoV-2 S2 subunit, fused in tandem.Subsequently, the protein antigen was expressed using insect cells and the Bac-to-Bac baculovirus expression system, as previously outlined. 14,15Following purification, we successfully obtained the RBD XBB.1.16-HR protein with over 98% purity and good homogeneity.Its formation was verified through gel-filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as depicted in Figure 1B.Additionally, we measured the binding interaction between our protein and human ACE2, which serves as the cellular receptor for SARS-CoV-2, through surface plasmon resonance (SPR) analysis.As anticipated, the kinetic binding data revealed an equilibrium dissociation constant (K D ) of 0.1748 nM (Figure 1C), coinciding with our prior findings and those of others. 16Additionally, negativestaining transmission electron microscopy (TEM) images of negative-stained samples confirmed the assembly of the protein into a trimeric structure, akin to the protein assembly mode depicted in the panel on the right (Figure 1D).

RBD XBB.1.16 -HR vaccine elicited robust cellular immune responses in mice spleen
We analyzed T cell and B cell responses in the spleens of vaccinated animals on day 70 (28 days after the third dose) using flow cytometry.Consistent with the previous studies, 14,17 the RBD XBB.1.16-HR vaccine exhibited robust germinal center (GC) responses, characterized by elevated levels of RBD (XBB.1.16)-specificmemory B cells (MBCs) and RBD-specific GC B cells and follicular helper T (Tfh) cells (Figure 3A-C).Regarding T cell responses, we assessed the populations of activated T cells, central memory T cells, and effector memory T cells via flow cytometry.Furthermore, we analyzed RBD-specific IFNγ-secreting T cells using an intracellular cytokine staining (ICS) assay.Our finding revealed increased CD4 + activated T cells in the RBD XBB.1.16-HR vaccine group (Figure 3D).Moreover, the frequencies of CD8 + and CD4 + central memory T cells were significantly elevated compared to the control group (Figure 3E,F), along with increased levels of CD4 + effector memory T cells (Figure 3G).The ICS assay demonstrated a significant increase in IFN-γsecreting CD8 + cells in the RBD XBB.1.16-HR vaccine group (Figure 3H).These results collectively indicated that the RBD XBB.1.16-HR vaccine could elicit a well-regulated GC center immune response and T cell immune response, which may persist over time and contribute to establishing memory immune responses.

RBD XBB.1.16 -HR heterologous booster improves humoral immune responses against XBB lineages
To assess the potential of the RBD XBB.1.16-HR booster vaccine in aged models (mouse >18 months), 18 we conducted a study using NIH female mice that had received a three-dose Delta-mRNA vaccine, followed by administration of the fourth booster or no booster (Figure 4A).

XBB.1.16 heterologous booster vaccine enhances the cellular immune response after priming three-dose Delta-mRNA vaccine
For cellular immune responses, three doses of Delta-mRNA vaccine induced strong frequencies of GC B cells and CD8 + CD44 + IFN-γ + in the vaccinated spleen for at least 2 years, while other cellular responses did not significantly differ from those in the PBS group.These findings underscore the need for a SARS-CoV-2 vaccine booster.In our study, the fourth dose of RBD XBB.1.16-HR vaccine effectively recalled and enhanced the priming memory immune responses, including MBCs (Figure 5A,B) and long-lived plasma cells (LLPCs) (Figure 5E).Although the frequency of Tfh cells did not significantly improve (Figure 5C,D), there was a notable elevation in RBDspecific GC B cells (Figure 5F).These data indicated that three-dose Delta-mRNA could induce a high level of central memory response for at least 2 years, and fourth-dose protein vaccine could activate the memory immune response.In terms of ICS, the frequency of CD4 + CD44 + IFN-γ + in the heterologous group was higher than in the Delta-mRNA group (Figure 5G), while the frequency of CD8 + CD44 + IFN-γ + cells was similar (Figure 5H).These results indicated that three doses of the Delta-mRNA vaccine could induce a comparable immune response against SARS-CoV-2 for at least 2 years.Moreover, a fourth RBD XBB.1.16-HR booster could reactivate Delta-mRNA-induced memory immune responses and enhance both humoral and cellular immune responses.

DISCUSSION
Considering the ongoing emergence of SARS-CoV-2 variants aimed at evading immune responses, updating COVID-19 vaccine compositions based on newly emerged viruses is necessary. 21Developing a recombinant protein vaccine based on XBB.1.16is crucial.In this study, we have developed a recombinant protein vaccine utilizing the RBD of the XBB.1.16variant and formulated it with an MF59-like adjuvant (RBD XBB.1.16-HR vaccine), as previously described. 14As expected, the RBD XBB.In a previous study, we demonstrated that following two-dose priming with the Delta-mRNA vaccine, a third heterologous RBD-HR (Delta) vaccine booster 3 months apart could induce more robust immune responses than a third homologous Delta-mRNA vaccine. 22In this study, we administrate the fourth booster (RBD XBB.1.16-HR vaccine or PBS) 2 years after the third-dose Delta-mRNA vaccine.Our data that doses of Delta-mRNA induce a comparable humoral immune response and cellular immune response.The fourth RBD XBB.1.16-HR booster significantly stimulated the humoral and cellular immune responses.
Compared with Delta mutant strains, BA.4/5-included Omicron sublineages have more substantial immune evasion capabilities. 23Fortunately, a bivalent BA.4/5-based mRNA vaccine (mRNA1273.222)has been authorized for booster vaccine use.Similar to mRNA1273.222, 9our data also showed that four doses of BA.4/5-mRNA vaccine induced potent neutralizing antibodies against XBB.1.5,XBB.1.6,XBB.1.16,XBB.1.16.6, XBB.2.3, and EG.5.1 pseudoviruses.However, a fourth booster vaccine, RBD XBB.1.16-HR, elicited more excellent broad-spectrum neutralizing antibodies than the homologous BA.4/5 mRNA vaccine.However, we acknowledge that our study did not analyze the difference in immunogenicity between the BA.4/5-mRNA vaccine induced by our method and other mRNA vaccines commercially available.These aspects represent important considerations for future research to comprehensively evaluate the effectiveness and comparative immunogenicity of different vaccine formulations and priming strategies in combating emerging SARS-CoV-2 variants.Such investigations would contribute valuable insights to optimize vaccination strategies and enhance global efforts to control the COVID-19 pandemic.
In conclusion, developing vaccines targeting newly emerged SARS-CoV-2 variants, such as the XBB.1.16subvariant, is crucial in the ongoing battle against the COVID-19 pandemic.Our study the potential of the recombinant XBB.1.16-HR vaccine to induce robust humoral and cellular immune responses against XBB sublineages, including those with increased immune evasion capabilities.Furthermore, our findings suggest that incorporating this vaccine as a booster shot could enhance immunity in individuals who have received previous doses of mRNA vaccines.While further research is needed to fully understand the efficacy and safety of the RBD XBB.1.16-HR vaccine in human populations, our study provides promising evidence for its potential role in combating the evolving landscape of SARS-CoV-2 variants.

Protein expression and characterization
The RBD XBB.1.16-HR protein was expressed in Spodoptera frugiperda Sf9 cells by Bac-to-Bac baculovirus expression system (Invitrogen) as previously described. 24Briefly, our construct design involved directly linking the S-RBD sequence of the SARS-CoV-2 XBB.1.16strain (amino acids 320-545) to sequences for HR1 and HR2 of the SARS-CoV-2 S2 subunit.For protein production, the coding sequences including a GP67 signal peptide, a Trx tag, a 6xHis tag, and an Enterokinase (EK) cleavage site were further fused into the N-terminus for protein secretion, folding, purification, and tag removal, respectively. 25hese gene fragments were initially amplified and then subcloned into pFastbac1 vector.Each sequencing-verified recombinant plasmid was transformed into Escherichia coli DH10b cells to produce recombinant bacmids, which were subsequently transfected into sf9 cells for protein production.[28][29]

Vaccine formulation
We formulated the RBD XBB.1.16-HR as previously. 14Briefly, the purified RBD XBB.1.16-HR protein was diluted in the MF59-like adjuvants in equal volumes to form vaccines with 200 µg/mL protein.
Delta-mRNA vaccines were formulated as before. 22The formulation of the BA.4/5-mRNA vaccine was similar to the Delta-mRNA vaccine unless the BA.4/5-mRNA vaccine expressed the full length of the spike protein of the BA.4/5 variant.

Mouse vaccination
The female NIH mice (6-8 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and maintained in the animal center of the State Key Laboratory of Biotherapy, Sichuan University.
To explore the immunogenicity of RBD XBB.1.16-HR vaccines, NIH mice were intramuscularly immunized with PBS, purified RBD XBB.1.16-HR recombinant protein, or RBD XBB.1.16-HR vaccination on days 0, 21, and 42.The vaccinated serum samples were collected on day 14, 35, and 56.To assay the cellular immune response induced by the RBD -HR vaccine, the of were collected and analyzed by To evaluate the potential of the RBD XBB.1.16-HR vaccine as a heterologous booster, the NIH mice (6-8 weeks) were intramuscularly immunized with two kinds of mRNA vaccines on day 0, 21, and 42. mRNA vaccine expresses the full-length S protein of the BA.4/5 variant.After three doses of BA.4/5-mRNA vaccine, a fourth homologous BA.5-mRNA vaccine (5 µg) or a heterologous RBD XBB.1.16-HR vaccine (10 µg) were immunized on day 100, serum and spleen were collected 14 days later after the fourth vaccination.

Enzyme-linked immunosorbent assays
Purified recombinant trimeric protein RBD XBB.1.16-HR was diluted to 1 µg/mL in carbonate coating buffer (50 mM, pH 9.6) and coated onto a 96-well plate (NUNC-MaxiSorp, Thermo Fisher Scientific) overnight (100 µL/well).The plate was then washed three times with PBS containing 0.1% Tween-20 (PBST) and blocked with 200 µL PBST containing 1% bovine serum albumin (BSA) for 1 h at room temperature.After washed, the plates were added with serially diluted sera (100 µL) and coated at room temperature.An hour later, the plates were washed with PBST more than three times and coated with diluted horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibodies (Invitrogen, 31430) for an hour at 37 • C. Following five washes with PBST, the plates were incubated with 100 µL of 3,3′,5,5′-tetramethylbenzidine (TMB, Thermo Fisher Scientific, 34029) per well for 10 min in the dark.The reaction was stopped by adding 100 µL/well Stop Solution for TMB Substrate (Beyotime, P0215), and the absorbance at 450 nm was measured using a microplate reader (Spectramax ABS, Molecular Devices) equipped with SoftMax Pro 7.1 software.

Pseudovirus neutralization assay
Several newly emerged pseudoviruses (Genomeditech) were used to explore RBD XBB.1.16-HR vaccine-induced broadly neutralizing antibodies.Briefly, threefold diluted sera (50 µL) were coincubated for an hour with 50 µL of diluted pseudovirus.Then, 1.2 × 10 4 293T/ACE2 cells (100 µL) were added to each well and incubated for 48 h.Wells without serum were seen as a negative control, and wells without pseudoviruses were seen as a positive control.Remove the supernatant and add 100 µL lysis reagent with luciferase substrate (Beyotime, RG005) to each well.The relative light unit (RLU) was assessed using a multimode microplate reader (PerkinElmer) with Kaleido 3.0 software, and analysis was conducted using GraphPad Prism 9.0. 14

Live SARS-CoV-2 virus neutralization assay
An authentic virus neutralization assay was established to detect vaccinated sera's neutralizing activity and inhibitory ability against emerged VOCs.In short, diluted serum samples were coincubated with live SARS-CoV-2 virus at 50% tissue-culture infectious dose (TCID50) for an hour at 37 • C before adding them to Vero cells (ATCC CCL-81) preplated at a density of 5 × 10 4 cells/well in 96-well culture plates.After 72 h, using a microscope, the neutralizing titers of vaccinated sera, resulting in 50% neutralization, were determined by observing cytopathogenic effects (CPEs).

Statistical analysis
Statistical analyses were conducted using Prism software (GraphPad Prism 8.0).Two-way analysis of variance (ANOVA) was employed to compare each cell mean with the other cell means in that row for two multivariate groups.The Mann-Whitney test was employed to compare two univariate groups.Comparisons involving three or more groups utilized one-way ANOVA followed by

C O N F L I C T O F I N T E R E S T S TAT E M E N T
This work was supported by the WestVac Biopharma Co. Ltd.Guangwen Lu and Xiawei Wei, who are also working at the WestVac Biopharma Co. Ltd.The remaining authors declare no conflicts of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
All the data from the corresponding authors are available upon reasonable request.

E T H I C S S TAT E M E N T
All animal studies followed and approved by the Institutional Animal Care and Use Committee of Sichuan University (Chengdu, Sichuan, China) with approval numbers at 20210409042 and 20230307025.

2
Receptor-binding domain (RBD) XBB.1.16-HR-induced humoral immune responses in serum.(A) Scheme of experiments.Six to eight-week-old NIH female mice were immunized with the RBD XBB.1.16-HR vaccine via an intramuscular route on days 0, 21, and 42.Sera samples were collected on days 14, 35, and 56.Serum on day 56 was used to measure the pseudovirus neutralization test and virus neutralization test (VNT).Immunized mice were excused on day 70, and the humoral and cellular immunization responses of the spleen were detected.(B-D) Humoral immunity response in serum of immunized mice.RBD-specific immunoglobulin G (IgG) antibodies (B) and neutralizing antibodies against pseudovirus (C) and authentic variants of concern (VOCs) (D).(A) was created by BioRender, (B-D) were prepared by GraphPad 9.0.Bars and columns show geometric mean with geometric standard deviation (SD) values.p values in (B) were determined by two-way analysis of variance (ANOVA) with compare each cell mean with the other cell mean in that row.*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.
Tukey's multiple comparisons test.*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.A U T H O R C O N T R I B U T I O N SXiawei Wei, Guangwen Lu conceived and supervised, and designed the experiments.Xiangrong Song performed the Delta-mRNA and BA.4/5-mRNA vaccine.Dandan Peng, Cai He, and Zimin Chen performed protein vaccine formu-lation and vaccinations in animals.Dandan Peng, Cai He, Xiya Huang, and Xinyi Du performed a binding antibodies assay and pseudovirus neutralization experiment.Dandan Peng, Cai He, Hong Lei, Xiya Huang, Chunjun Ye, Binhan Wang, and Ying Hao collected tissues and performed flow cytometry to assay cellular immune responses.Dandan Peng and Cai He analyzed the data and wrote the manuscript.All the authors have read and approved the final manuscript.A C K N O W L E D G M E N T S This work was supported by the National Science Foundation for Excellent Young Scholars (No. 32122052, Xiawei Wei), National Natural Science Foundation Regional Innovation and Development (No. U19A2003, Xiawei Wei), and National Natural Science Foundation of China Youth Fund (No. 32300743).Figures 2A and 4A,B were created by BioRender.